This invention relates to a cell membrane channel responsible for the so-called calcium conductance observed in neuronal and other cell membranes. This invention also relates to methods for purifying this channel and to factors and methods for regulating or blocking calcium conductance in cellular membranes.
Passive transport of charged particles across cell membranes, in response to an incremental change in an electrical field across the thickness of the cell membrane, is mediated (and, in substantial part, regulated) by membrane channel proteins.
In this discussion the terms "channel" and "channel protein" are used interchangeably without implying that a channel must necessarily consist of a single protein, although the channels which have been isolated are believed to be single proteins.
Most channel proteins are believed to mediate the transport of one ionic species with substantially higher specificity than transport of other ions. Although the existence of several channels has been experimentally demonstrated, fewer than ten channel proteins have been isolated. These include certain sodium and potassium channel proteins, the isolation of which has been described in the U.S. patent application Ser. Nos. 948,262 filed on Dec. 31, 1986 and 085,462 filed on Aug. 17, 1987 now abandoned both in the name of Bruce Cherksey the disclosures of which are incorporated by reference in their entirety.
Calcium channels have been shown to be responsible for highthreshold calcium conductance (HTCC) observed in responses to direct electric impulse (or synaptic) stimulation of neurons. This conductance is responsible for the calcium-dependent action potentials, especially in the dendrites (Llinas, R. and Sugimori, M. J. Physiol., 305: 197-213, 1980). Calcium channels have also been shown to be responsible for a low-threshold calcium conductance (LTCC) which generates calcium-dependent spikes from a rather negative value of membrane potential (-65 mV). This LTCC spike often appears as a rebound depolarization following the after hyperpolarization potential which in turn follows the after depolarization potential due to the HTCC (Llinas and Yarom, J. Physiol., 315: 549-567 and 569-584, 1981) Calcium channels are also involved in presynaptic transmitter release during synaptic transmission. Other cell types which possess such calcium channels include heart muscle fibers and endocrine cells.
Before the present invention, calcium channels were known to be structures spanning the lipid bilayer of the cell membrane and demonstrating high (though not exclusive) specificity for the transport of calcium ions through this membrane. Despite being the subject of considerable research effort, the types of calcium channel structures responsible for central neuron spike activity had not been isolated nor identified.
Among the reasons for the failure to isolate calcium channels was the unavailability of a material having at least one of the following properties:
ability to bind calcium channels specifically, tightly (with high affinity) and reversibly and to block the calcium conductance completely; and
ability to be labelled by a fluorescent or other detectable marker while retaining the ability to bind the calcium channel thus making it possible to identify the location and quantify the occurrence of calcium channels on a cell membrane.
Previously known calcium channel blocking proteins such as nitrendipene, D600 (methoxyverapamil), doxorubicin hydrochloride, and quinidine could not be used for identification of the calcium channel because they bind the channel either nonspecifically or irreversibly or both. Also, some of these agents, notably dihydropyridines, do not recognize the type of channels responsible for calcium conductance in cerebellar neurons on which the experiments illustrating the present invention were conducted: R. J. Miller, infra.
Various natural toxins have recently become the focus of attention as potential tools for studying neuronal channels.
In the experience of the present inventors, conotoxin (a toxin from the venom of the marine snail Conus geographicus) which has been reported to block calcium channels (Miller, R. J., Science, 235:46, 1987) does not bind the calcium channels under investigation with sufficient affinity to be useful for channel isolation and purification.
Toxins present in or extracted from the venom of funnel-web spiders have also been the subject of substantial investigation. Sheumack, D. D., et al., FEBS 2237, 181:154 (1985) report the sequencing of a polypeptide toxin from the funnel-web spider Atrax robustus. The sequence of this polypeptide is said to contain 42 amino acid residues including several disulphide-bridged cysteine residues.
Venom and several chromatographic extracts from the venom of the Agelenopsis aperta spider, a common funnel-web spider indigenous to the continental United States, have also been under study.
A 6000 dalton molecular weight toxin derived from A.aperta venom was said to block synaptic transmission in chick brain stem neurons in a manner dependent on the extracellular calcium ion concentration: H. Jackson et al Society for Neuroscience, Abstracts 16th Annual Meeting, Washington, D.C. Nov. 9-14, 1986. The authors raise the possibility that the toxin might block either calcium channels or the synaptic release process itself and stated that the binding of the toxin appeared to be "very tight if not irreversible".
The same group of investigators have studied toxins from other spiders including the funnel-web spider Hololena curta and reported that one such toxin (estimated mw 5,000-10,000 daltons) blocks postsynaptic responses irreversibly. Another toxin said to be derived from A.aperta venom is also reported to irreversibly block transmission in a manner dependent on the extracellular calcium ion concentration. Jackson, H. et al. in Excitatory Amino Acid Transmission, pp. 51-54 (Alan R. Liss, Inc., New York 1987).
Bowers, C. W., et al., PNAS (U.S.A.) 84: 3506 (1987), report that a toxin isolated from Hololena curta appears to have a specific and direct effect on presynaptic calcium channels in neurons. The toxin is said to be a polypeptide composed of at least two disulphide-linked subunits of apparent molecular weights of 7000 and 9000 based on SDS-PAGE (sodium dodecyl sulfate electrophoresis). The authors hypothesize that this toxin acts by a potent and long-lasting inhibition of voltage-dependent presynaptic calcium channels and propose its use as a molecular probe for synaptic physiology.
Adams, M. E., et al, Insect Neurochem. Neurophysiol. (Pap. Int. Conf.) 2d, 397-400, 1986, report that they have isolated several toxins from A.aperta venom. One group of toxins are said to be polypeptides having an apparent molecular weight of 4800 daltons. Partial sequence information confirmed the polypeptide nature of these toxins and indicated strong homology among them. These toxins were not inactivated by boiling and three among them were resistant to trypsin. The authors stated that the presence of multiple cysteine residues within the sequences of these toxins raised the possibility that the structure of these toxins would have several disulphide bridges. Their activity is attributed to a presynaptic action on the sodium channel which was not reversed even after hours of washing.
Another smaller toxin (of molecular weight said to be less than 1000) isolated by the same investigators is said to be hydrophylic and to act postsynaptically because it was observed to cause a gradual diminution of the excitatory postsynaptic potential (EPSP) leading to its eventual block. However, this toxin is not otherwise characterized and the method for isolating it from A.aperta venom is not described. (In contrast, as will be shown below, the active factors of the present invention have a molecular weight of 300-500 daltons and act presynaptically.)
The authors of Adams et al, suora, also raised the possibility of using these toxins as pharmacological tools in the identification of chemicals affecting synaptic transmission.
Isolation and identification of calcium channels is of considerable interest because it would provide methods for regulating calcium ion transport through cell membranes (notably via use of appropriate blocking agents) which would have several research and diagnostic applications as well as therapeutic potential. Identification of such channels would also help increase the scientific understanding of membrane transport mechanisms.
Novel methods and factors for specifically and reversibly blocking calcium channels with high affinity would be useful, inter alia, in isolation and identification of calcium channels, in selective blocking of such channels (to avoid interference due to calcium channel-mediated responses with other membrane phenomena under study) and in drug screening and design.